The glutathione-dependent system is one of the key systems regulating cellular redox balance, and thus cell fate. Cysteine, typically present in its oxidized form cystine in the extracellular space, is regarded as the rate-limiting substrate for glutathione (GSH) synthesis. Cystine is transported into cells by the highly specific amino-acid antiporter system x c À . Since Burkitt's Lymphoma (BL) cells display limited uptake capacity for cystine, and are thus prone to oxidative stress-induced cell death, we stably expressed the substrate-specific subunit of system x c À , xCT, in HH514 BL cells. xCT-overexpressing cells became highly resistant to oxidative stress, particularly upon GSH depletion. Contrary to previous predictions, the increase of intracellular cysteine did not affect the cellular GSH pool, but concomitantly boosted extracellular cysteine concentrations. Even though cells were depleted of bulk GSH, xCT overexpression maintained cellular integrity by protecting against lipid peroxidation, a very early event in cell death progression. Our results show that system x c À protects against oxidative stress not by elevating intracellular GSH levels, but rather creates a reducing extracellular environment by driving a highly efficient cystine/cysteine redox cycle. Our findings show that the cystine/cysteine redox cycle by itself must be viewed as a discrete major regulator of cell survival.

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/ reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in ␥-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.

Tumor cells generate substantial amounts of reactive oxygen species (ROS), engendering the need to maintain high levels of antioxidants such as thioredoxin (Trx)-and glutathione (GSH)-dependent enzymes. Exacerbating oxidative stress by specifically inhibiting these types of ROS-scavenging enzymes has emerged as a promising chemotherapeutic strategy to kill tumor cells. However, potential redundancies among the various antioxidant systems may constrain this simple approach. Trx1 and thioredoxin reductase 1 (Txnrd1) are upregulated in numerous cancers, and Txnrd1 has been reported to be indispensable for tumorigenesis. However, we report here that genetic ablation of Txnrd1 has no apparent effect on tumor cell behavior based on similar proliferative, clonogenic, and tumorigenic potential. This finding reflects widespread redundancies between the Trx-and GSH-dependent systems based on evidence of a bypass to Txnrd1 deficiency by compensatory upregulation of GSH-metabolizing enzymes. Because the survival and growth of Txnrd1-deficient tumors were strictly dependent on a functional GSH system, Txnrd1 −/− tumors were highly susceptible to experimental GSH depletion in vitro and in vivo. Thus, our findings establish for the first time that a concomitant inhibition of the two major antioxidant systems is highly effective in killing tumor, highlighting a promising strategy to combat cancer. Cancer Res; 70(22); 9505-14. ©2010 AACR.

IntroductionFeeding a Bones and Raw Food (BARF) diet has become an increasing trend in canine nutrition. Bones and Raw Food diets contain a high amount of animal components like meat, offal, and raw meaty bones, combined with comparatively small amounts of plant ingredients like vegetables and fruits as well as different sorts of oil and supplements. While many studies have focused on transmission of pathogens via contaminated meat and on nutritional imbalances, only few studies have evaluated the effect of BARF diets on the fecal microbiome and metabolome. The aim of the study was to investigate differences in the fecal microbiome and the metabolome between dogs on a BARF diet and dogs on a commercial diet (canned and dry dog food).MethodsNaturally passed fecal samples were obtained from 27 BARF and 19 commercially fed dogs. Differences in crude protein, fat, fiber, and NFE (Nitrogen-Free Extract) between diets were calculated with a scientific nutrient database. The fecal microbiota was analyzed by 16S rRNA gene sequencing and quantitative PCR assays. The fecal metabolome was analyzed in 10 BARF and 9 commercially fed dogs via untargeted metabolomics approach.ResultsDogs in the BARF group were fed a significantly higher amount of protein and fat and significantly lower amount of NFE and fiber. There was no significant difference in alpha-diversity measures between diet groups. Analysis of similarity (ANOSIM) revealed a significant difference in beta-diversity (p < 0.01) between both groups. Linear discriminant analysis effect size (LefSe) showed a higher abundance of Lactobacillales, Enterobacteriaceae, Fusobacterium and, Clostridium in the BARF group while conventionally fed dogs had a higher abundance of Clostridiaceae, Erysipelotrichaceae, Ruminococcaceae, and Lachnospiraceae. The qPCR assays revealed significantly higher abundance of Escherichia coli (E. coli) and Clostridium (C.). perfringens and an increased Dysbiosis Index in the BARF group. Principal component analysis (PCA) plots of metabolomics data showed clustering between diet groups. Random forest analysis showed differences in the abundance of various components, including increased 4-hydroxybutryric acid (GBH) and 4-aminobutyric acid (GABA) in the BARF group. Based on univariate statistics, several metabolites were significantly different between diet groups, but lost significance after adjusting for multiple comparison. No differences were found in fecal bile acid concentrations, but the BARF group had a higher fecal concentration of cholesterol in their feces compared to conventionally fed dogs.ConclusionMicrobial communities and metabolome vary significantly between BARF and commercially fed dogs.

The photoinduced ring-opening reaction of cyclohexadiene (CHD) is a textbook example for electrocyclic reactions. In this paper we report the complete "minimum energy path" of the low-lying region of the conical intersection space reaching from the closed to the open ring side. The general role of conical intersections (CoIns) is to provide the locus for ultrafast transfer between electronic states, in the present case, to the closed or open form in the ground state after photoexcitation. The seam was calculated with use of an analytic approach in which the intersection space in the vicinity of a CoIn is described to second order. The topography of the seam was investigated, revealing minimum energy and transition state structures. In addition the energy profile of the seam was rationalized with valence bond (VB) theory. The geometrical changes along the seam have been related to the motions along the excited state minimum energy path (S(1)-IRC-MEP) in a conceptual model highlighting the quasiparallel orientation of seam and IRC-MEP. Our model shows that even though the van der Lugt and Oosterhoff concept predicts the formation of an avoided crossing along the S(1)-IRC-MEP, it provides an incomplete description of the decay process to the ground state. The latter requires, in addition, vibrational motions orthogonal to the MEP, directed toward the CoIn seam.

Rationale: Growing evidence indicates that oxidative stress contributes markedly to endothelial dysfunction. The selenoenzyme glutathione peroxidase 4 (Gpx4) is an intracellular antioxidant enzyme important for the protection of membranes by its unique activity to reduce complex hydroperoxides in membrane bilayers and lipoprotein particles. Yet a role of Gpx4 in endothelial cell function has remained enigmatic. Objective: To investigate the role of Gpx4 ablation and subsequent lipid peroxidation in the vascular compartment in vivo. Methods and Results: Endothelium-specific deletion of Gpx4 had no obvious impact on normal vascular homeostasis, nor did it impair tumor-derived angiogenesis in mice maintained on a normal diet. In stark contrast, aortic explants from endothelium-specific Gpx4 knockout mice showed a markedly reduced number of endothelial branches in sprouting assays. To shed light onto this apparent discrepancy between the in vivo and ex vivo results, we depleted mice of a second antioxidant, vitamin E, which is normally absent under ex vivo conditions. Therefore, mice were fed a vitamin E–depleted diet for 6 weeks before endothelial deletion of Gpx4 was induced by 4-hydroxytamoxifen. Surprisingly, ≈80% of the knockout mice died. Histopathological analysis revealed detachment of endothelial cells from the basement membrane and endothelial cell death in multiple organs, which triggered thrombus formation. Thromboembolic events were the likely cause of various clinical pathologies, including heart failure, renal and splenic microinfarctions, and paraplegia. Conclusions: Here, we show for the first time that in the absence of Gpx4, sufficient vitamin E supplementation is crucial for endothelial viability.

The fast and slow components of the relaxation of photoexcited thiophene have been investigated by means of SHARC (surface hopping including arbitrary couplings) molecular dynamics based on multiconfiguration electronic structure calculations. Triplet states are included to ascertain their role in the relaxation process. After thiophene is excited to the S state, ultrafast dynamics (τ = 96 fs) initiates a ring opening due to cleavage of a carbon sulfur bond and simultaneous ring puckering. This time constant is in agreement with previous experimental and theoretical studies. The subsequent dynamics of the open-ring structures is characterized by the interplay of internal conversion and intersystem crossing. For the open-ring structures, the S, S, T and T states are nearly degenerate and the spin-orbit couplings are large. The underlying potential energy surface is flat and long-lived open-ring structures in the singlet as well as in the triplet states are formed. Both the participation of triplet states and the shape of the energy surface explain the experimentally observed slow ring closure in the ground state.

Significant differences in the reactivity of norbornene derivatives in the inverse electron-demand Diels-Alder reaction with tetrazines were revealed by kinetic studies. Substantial rate enhancement for the exo norbornene isomers was observed. Quantum-chemical calculations were used to rationalize and support the observed experimental data.